SIST EN 14663:2006

SLOVENSKI STANDARD
SIST EN 14663:2006
01-marec-2006
äLYLOD±'RORþHYDQMHYLWDPLQD%YNOMXþQR]JOLNRVLOLUDQRREOLNRV+3/&
Foodstuffs - Determination of vitamin B6 (including its glycosylated forms) by HPLC
Lebensmittel - Bestimmung von Vitamin B6 (einschließlich glucosidisch gebundener
Verbindungen) mit HPLC
Produits alimentaires - Dosage de la vitamine B6 (y compris ses formes glycosylées) par
CLHP
Ta slovenski standard je istoveten z: EN 14663:2005
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST EN 14663:2006 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 14663:2006

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SIST EN 14663:2006
EUROPEAN STANDARD
EN 14663
NORME EUROPÉENNE
EUROPÄISCHE NORM
December 2005
ICS 67.050

English Version
Foodstuffs - Determination of vitamin B6 (including its
glycosylated forms) by HPLC
Produits alimentaires - Dosage de la vitamine B6 (y Lebensmittel - Bestimmung von Vitamin B6 (einschließlich
compris ses formes glycosylées) par CLHP glucosidisch gebundener Verbindungen) mit HPLC
This European Standard was approved by CEN on 26 October 2005.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the Central Secretariat or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,
Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2005 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 14663:2005: E
worldwide for CEN national Members.

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SIST EN 14663:2006
EN 14663:2005 (E)
Contents Page
Foreword .3
1 Scope.4
2 Normative references.4
3 Principle.4
4 Reagents.4
5 Apparatus.9
6 Procedure.9
7 Calculation.11
8 Precision.12
9 Test report.14
Annex A (informative)  Precision data .15
Annex B (informative) Examples for suitable HPLC-conditions for the determination of vitamin B
6
compounds.19
Annex C (informative)  Examples for molar extinction coefficients .20
Annex D (informative) Figures.21
Bibliography.22

Figure
Figure D.1 — Standard substances and sample potato puree.21

Tables
Table 1 — Examples for molecular extinction coefficients of vitamin B6 compounds.7
Table A.1 — Precision data for Semolina with milk, powder.15
Table A.2 — Precision data for Potato puree,powder.16
Table A.3 — Precision data for vegetables with ham (baby food) .17
Table A.4 — Precision data for multi vitamin drink.18
Table B.1 — Examples for suitable HPLC-conditions for the determination of vitamin B6
compounds .19
Table C.1 — Examples for molar extinction coefficients (E) of vitamin B6 compounds [3], [4] .20


2

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SIST EN 14663:2006
EN 14663:2005 (E)
Foreword
This document (EN 14663:2005) has been prepared by Technical Committee CEN/TC 275 “Food analysis -
Horizontal methods”, the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by June 2006, and conflicting national standards shall be withdrawn at
the latest by June 2006.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic,
Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland
and United Kingdom.
3

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SIST EN 14663:2006
EN 14663:2005 (E)
1 Scope
This document specifies a method for the determination of vitamin B in foodstuffs by high performance liquid
6
chromatography (HPLC).
Vitamin B is the mass fraction of the sum of pyridoxine, pyridoxal, pyridoxamine including their
6
phosphorylated derivatives as well as the β-glycosylated forms, calculated as pyridoxine.
This method has been successfully validated with semolina with milk (infant food), potato puree, vegetables
with ham (convenient products) and a multi vitamin drink at levels from 0,034 mg/100 g to 1,21 mg/100 g.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
EN ISO 3696, Water for analytical laboratory use — Specification and test methods (ISO 3696:1987).
3 Principle
Pyridoxal, pyridoxamine and pyridoxine are extracted from food by acid hydrolysis and dephosphorylated and
deglycosilated enzymatically using acid phosphatase and β-glucosidase.
The different derivatives of vitamin B (pyridoxal, pyridoxamine and pyridoxine) are separated by HPLC and
6
quantified by fluorometric detection [1], [2].
4 Reagents
4.1 General
During the analysis, unless otherwise stated, use only reagents of recognised analytical grade and water of at
least grade 1 according to EN ISO 3696, or double distilled water.
4.2 Di-potassium hydrogen phosphate, mass fraction w(K HPO · 3 H O) ≥ 99,9 %
2 4 2
4.3 Sodium acetate, without crystal water, w(CH COONa) ≥ 99,0 %
3
4.4 Trichloroacetic acid (TCA), w(Cl CCOOH) ≥ 99,0 %
3
4.5 Sodium acetate solution, substance concentration c(CH COONa) = 2,5 mol/l
3
Dissolve 205 g of sodium acetate (4.3) in 1 l of water.
4.6 Post-column reagent (optional), K HPO solution c(K HPO ) = 0,15 mol/l
2 4 2 4
Dissolve 34,2 g of di-potassium hydrogen phosphate (4.2) in water, dilute to 1 000 ml, mix and degas.
4

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SIST EN 14663:2006
EN 14663:2005 (E)
4.7 Hydrochloric acid, c(HCl) = 1 mol/l
c(HCl) = 0,1 mol/l
4.8 Hydrochloric acid,
4.9 Hydrochloric acid, c(HCl) = 0,2 mol/l
4.10 Sulfuric acid, c(H SO )= 1 mol/l
2 4
4.11 Light petroleum, boiling range of 40 °C to 60 °C
1)
4.12 Acid phosphatase, from potatoes. Enzymatic activity approximately 5,3 U/mg .
It is important that the enzyme used complies with the activity check 4.13.2, for further information see [2], [7].
4.13 Acid phosphatase solution
4.13.1 General
Dissolve/solubilise 60 mg of acidic phosphatase (4.12) in 10 ml of water in a beaker by stirring for 2 min.
Prepare this solution on the day of analysis.
4.13.2 Activity check of Acid Phosphatase
Weigh 10 g of pork, 5 g of potato puree or 5 g of whole meal into a beaker, and extract with acid as described
in 6.2.1. Add 1 ml of acid phosphatase solution (4.13.1) and optional 1 ml of β-glucosidase solution (4.15) to
12,5 ml of the extracted sample solution and mix. Incubate the solution at least 12 h or overnight at 37 °C with
continuous stirring. Repeat this step with the double amount of acid phosphatase solution.
Determine the mass concentration of vitamins according to 6.6. The activity of the enzyme used is sufficient, if
the resulting mass concentrations of vitamin B compounds in both sample solutions are equivalent. The
6
chromatogram shall not show a peak arising from pyridoxamin phosphate.
)
2
NOTE For the interlaboratory test, the acid phosphatase from Sigma Nr P 3752 has been used.
4.14 ββ-Glucosidase, from almonds. Enzymatic activity of approximately 3,2 U/mg.
ββ
It is important that the enzyme used complies with the activity check 4.15.2, for further information see [2], [7].
4.15 ββββ-Glucosidase solution
4.15.1 General
Dissolve/solubilise 100 mg of β-glucosidase (4.14) in 10 ml of water in a beaker by stirring for 2 min. Prepare
this solution on the day of analysis.

1)
U, this unit (often called the International unit or standard unit) is defined as the amount of enzyme which catalyses
the transformation of 1 µmol substrate per minute under standard conditions.
2
) This information is given for the convenience of users of this document and does not constitute an endorsement by
CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results.
5

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SIST EN 14663:2006
EN 14663:2005 (E)
4.15.2 Activity check of ββ-glucosidase
ββ
Weigh 10 g of pork, 5 g of potato puree or 5 g of whole meal into a beaker, and extract with acid as described
in 6.2.1. Add 1 ml of acid phosphatase solution (4.13.1) and 1 ml of β-glucosidase solution (4.15.1) to 12,5 ml
of the extracted sample solution and mix. Incubate the solution at least 12 h or overnight at 37 °C with
continuous stirring. Repeat this step with the double amount β-glucosidase solution.
Determine the mass concentration of vitamin B compounds according to 6.6. The activity of the enzyme used
6
is sufficient, if the resulting mass concentrations of vitamin B compounds in both sample solutions are
6
equivalent. The chromatogram shall not show a peak arising from pyridoxamin phosphate.
)
1
NOTE For the interlaboratory test, the β-glucosidase from Sigma Nr G-0395 has been used.
4.16 Mobile phase for HPLC (Sulfuric acid, c(H SO )= 0,015 mol/l containing 0,005 mol/l TCA)
2 4
Dissolve 817 mg ± 5 mg of trichloroacetic acid (4.4) in 15 ml of 1 mol/l sulfuric acid (4.10), transfer into a 1 000 ml
volumetric flask, dilute to the mark with water, mix and degas.
4.17 Silicon oil, for defoaming
4.18 Standard substances
4.18.1 General
Pyridoxamine (PM), Pyridoxal (PL) and pyridoxine (PN) can be obtained from various suppliers. The purity of
the standards may vary, and it is therefore necessary to determine the concentration and purity (see 4.19.4
and 4.20.7).
4.18.2 Pyridoxamine (PM) dihydrochloride, w(C H N O · 2HCl) ≥ 98 %
8 12 2 2
4.18.3 Pyridoxal (PL) hydrochloride, w(C H NO · HCl) ≥ 98 %
8 9 3
4.18.4 Pyridoxine (PN) hydrochloride, w(C H NO · HCl ) ≥ 98 %
8 11 3
4.19 Stock solutions
4.19.1 Pyridoxamine (PM) stock solution, mass concentration ρ(PM) approximately 500 µg/ml
Dissolve 71,7 mg of pyridoxamine dihydrochloride (4.18.2) in a 100 ml volumetric flask in 0,1 mol/l HCl (4.8) and
dilute to the mark with 0,1 mol/l HCl. The solution can be stored without any losses for up to one week at 4 °C or
up to two months at -18 °C.
4.19.2 Pyridoxal (PL) stock solution, ρ(PL) approximately 500 µg/ml
Dissolve 60,9 mg of pyridoxal hydrochloride (4.18.3) in a 100 ml volumetric flask in 0,1 mol/l HCl (4.8) and dilute
to the mark with 0,1 mol/l HCl. The solution can be stored without any losses for up to one week at 4 °C or up to
two months at -18 °C.

1)
This information is given for the convenience of users of this document and does not constitute an endorsement by
CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results.
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SIST EN 14663:2006
EN 14663:2005 (E)
4.19.3 Pyridoxine (PN) stock solution, ρ(PN) approximately 500 µg/ml
Dissolve 60,8 mg of pyridoxine hydrochloride (4.18.4) in a 100 ml volumetric flask in 0,1 mol/l HCl (4.8) and dilute
to the mark with 0,1 mol/l HCl. The solution can be stored without any losses for up to one week at 4 °C or up to
two months at – 18 °C.
4.19.4 Concentration tests
Pipette 1 ml of stock solutions of pyridoxamine (4.19.1), pyridoxal (4.19.2) and pyridoxine (4.19.3) respectively in a
50 ml volumetric flask and dilute to the mark with 0,1 mol/l HCl (4.8). Measure the absorbance of the solutions in a
1 cm quartz-cell against 0,1 mol/l HCl at the maximum wavelength using UV-spectrometry (see Table 1).
Calculate the mass concentration of each vitamin B compound, ρ, using the molar extinction coefficient as
6 i
given in equation (1):
A×M
i
ρ = ×V ×F (1)
i
ε
i
where:
ρ is the mass concentration of pyridoxamine, pyridoxal and pyridoxine respectively in microgram per
i
millilitre stock solution;
A is the absorbance value of pyridoxamine, pyridoxal and pyridoxine solutions at the maximum
wavelength λ (see table 1);
max
ε is the molecular absorbance coefficient of PM, PL or PN at the appropriate pH as defined in table 1;
i
M is the molecular weight of PM, PL and PN respectively standard substances as defined in table 1;
i
V is the dilution factor, in this case V = 50;
F is the calculation factor of HCl free vitamin B compounds.
6
Use these mass concentrations to calculate the exact concentrations of 4.19.1 to 4.19.3 and 4.20.1 to 4.20.6.
Table 1 — Examples for molecular extinction coefficients of vitamin B compounds
6
Compounds Solvent λ M F
max i
ε
i
–1
-1 -1
g mol
mmol cm
.
a
0,1 mol/l HCl, pH ~1 292 8,2 241,1 0,698
PM 2 HCl
.
b
0,1 mol/l HCl, pH ~1 288 9,0 203,6 0,821
PL HCl
.
c
0,1 mol/l HCl, pH ~1 291 8,6 205,6 0,823
PN HCl
.
a
PM 2 HCl = Pyridoxamine-dihydrochloride (4.18.2)
.
b
PL HCl = Pyridoxal-hydrochloride (4.18.3)
.
c
PN HCl = Pyridoxine-hydrochloride (4.18.4)
4.20 Standard solutions
4.20.1 Pyridoxamine (PM) standard solution I, ρ(PM) approximately 10 µg/ml
Dilute 2 ml of pyridoxamine stock solution (4.19.1) with 0,1 mol/l HCl (4.8) to 100 ml. Prepare freshly every day
7

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SIST EN 14663:2006
EN 14663:2005 (E)
4.20.2 Pyridoxal (PL) standard solution I, ρ(PL) approximately 10 µg/ml
Dilute 2 ml of PL stock solution (4.19.2) with 0,1 mol/l HCl (4.8) to 100 ml. Prepare freshly every day
4.20.3 Pyridoxine (PN) standard solution I, ρ(PN) approximately 10 µg/ml
Dilute 2 ml of pyridoxine stock solution (4.19.3) with 0,1 mol/l HCl (4.8) to 100 ml. Prepare freshly every day
4.20.4 Pyridoxamine (PM) standard solution II, ρ(PM) approximately 1 µg/ml
Dilute 10 ml of standard solution I (4.20.1) with 0,1 mol/l HCl (4.8) to 100 ml. Prepare freshly every day
4.20.5 Pyridoxal (PL) standard solution II, ρ(PL) approximately 1 µg/ml
Dilute 10 ml of PL standard solution I (4.20.2) with 0,1 mol/l HCl (4.8) to 100 ml. Prepare freshly every day
4.20.6 Pyridoxine (PN)standard solution II, ρ(PN) approximately 1 µg/ml
Dilute 10 ml of pyridoxine standard solution I (4.20.3) with 0,1 mol/l HCl (4.8) to 100 ml. Prepare freshly every day
4.20.7 Check of chromatographic purity by HPLC
Purity of standard substances can be checked by HPLC as follows:
Inject appropriate volumes of PM, PL and PN standard solutions I (4.20.1, 4.20.2, 4.20.3) into the HPLC system
and analyse as described in 6.4.
Calculate purity of the standard substances according to equation (2):
x × 100
i
R = (2)
i
x + B
i
where
R is the purity of standard substance i in %;
i
x is the peak area of standard substance i;
i
B is the sum of the peak areas of contaminating substances (without solvent peak).
The chromatographic purity of standard substances should be ≥ 98 %, otherwise take new standard substances
or prepare new standard solutions.
4.21 Mixed calibration solution e. g. ρ(PM, PL, PN) = 0,1 µg/ml to 10 µg/ml
Pipette suitable volumes of PM, PL and PN stock solutions (4.19.1 to 4.19.3) or standard solutions (4.20.1 to
4.20.6) into a 20 ml volumetric flask, dilute with 0,1 mol/l HCl (4.8) to 6,5 ml, if necessary. Adjust to pH = 4,8 with
2,5 mol/l sodium acetate solution (4.5), and then adjust to pH = 3,0 with sulfuric acid (4.10), dilute with water to the
mark and mix (calibration solutions). At least three calibration points are recommended. If necessary, the mixed
calibration solutions may be diluted with mobile phase prior to HPLC injection.
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SIST EN 14663:2006
EN 14663:2005 (E)
5 Apparatus
5.1 General
Usual laboratory apparatus, glassware, and the following.
5.2 UV Spectrometer, capable of measurement of absorbance at defined wavelengths
5.3 Heating devices
Laboratory autoclave and oven or water bath, with stirring facilities, able to be controlled at 37 °C
5.4 High performance liquid chromatographic system
Consisting of a pump, sample injecting device, fluorescence detector with excitation and emission
wavelengths set at 290 nm and 390 nm, respectively and an evaluation system such as an integrator, and
optionally, a post column derivatisation device
5.5 HPLC-Column, e. g. reversed phase column, such as:
TM )
1 2)
Luna RP C , 5 µm , particle size of 5 µm, diameter 4,0 mm, length 250 mm . Other suitable examples are
18
listed in Annex B
5.6 Filter device
Filtering of the mobile phase as well as of the test sample solution through a membrane filter, with e. g. a pore
size of 0,45 µm, prior to use or injection will increase longevity of the columns
6 Procedure
6.1 Preparation of the test sample
Cut and homogenise the test sample. Grind coarse material with an appropriate mill and mix again. Measures
such as pre-cooling have to be taken to avoid exposing to high temperature for long periods of time. After
homogenising, analyse the sample immediately.
6.2 Preparation of the sample test solution
6.2.1 Extraction
6.2.1.1 General
For samples with a high fat content (> 25 %) it can be useful to remove fat e.g. by repeated treatment with
light petroleum before the acid hydrolysis.

TM
1)
Luna is an example of a commercially available product, supplied by Phenomenex. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of the product named. Equivalent
products may be used if they can be shown to lead to the same results.
2)
Other particle sizes or column dimensions than specified in this document may be used. Separation parameters have
to be adapted to such materials to guarantee equivalent results. The performance criterion for suitable analytical columns
is the baseline resolution of the analytes concerned.
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SIST EN 14663:2006
EN 14663:2005 (E)
For treatment of foaming material the use of few drops of silicon oil (4.17) is recommended.
The pH of the extracted solution should be approximately 1. Otherwise it is advisable to reduce the sample weight
or to use hydrochloric acid with higher concentration (e. g. 0,2 mol/l (4.9) or even 1 mol/l (4.7)).
6.2.1.2 Extraction of dry products (water content < 20 %, e. g. cereals, dried milk, dried vegetables)
Weigh 1 g to 10 g of the homogenised test sample (6.1) to nearest milligram into a 150 ml conical flask, add 50 ml
of 0,1 mol/l hydrochloride acid (4.8), mix and check that the pH is approximately 1.
Heat in
...